Vertebrates possess two distinct types of adipose tissue: white adipose tissue (WAT) and brown adipose tissue (BAT). WAT stores and releases fat according to the nutritional needs of the animal. BAT burns fat, releasing the energy as heat (i.e., nonshivering heat). The unique thermogenic properties of BAT reflect the activities of specialized mitochondria that contain the brown adipocyte-specific gene product uncoupling protein (UCP). Sears, I. B. et al. (1996) Mol. Cell. Biol. 16(7):3410-3419. UCP is a mitochondrial proton carrier that uncouples respiration from oxidative phosphorylation by collapsing the proton gradient established from fatty acid oxidation without concomitant ATP synthesis (Nicholls, D. and Locke, R. (1984) Physiol. Rev. 64:1-64).
UCP expression is tightly regulated, primarily by sympathetic nervous systems, in response to physiological signals, such as cold exposure and excess caloric intake (Girardier, L. and Seydoux, J. (1986) “Neural Control of Brown Adipose Tissue” In P. Trayhurn and D. Nichols (eds.) Brown Adipose Tissue (Arnold, London, 19 6) Pp. 122-151. Norepinephrine released from the local neurons interacts with β-adrenergic receptors on the brown adipocyte cell membrane, causing an increase in intracellular cyclic AMP (cAMP) levels (Sears, I. B. et al. (1996) Mol. Cell. Biol. 16(7):3410-3419). An increased level of transcription of the UCP gene is a critical component in the cascade of events leading to elevated BAT thermogenesis in response to increased cAMP (Kopecky, J. et al. (1990) J. Biol. Chem. 265:22204-22209; Rehnmark, S. M et al. (1990) J. Biol. Chem. 265:16464-16471; Ricquier, D. F. et al. (1986) J. Biol. Chem. 261:13905-13910). BAT thermogenesis is used both (1) to maintain homeothermy by increasing thermogenesis in response to lower temperatures and (2) to maintain energy balance by increasing energy expenditure in response to increases in caloric intake (Sears, I. B. et al. (1996) Mol. Cell. Biol. 16(7):3410-3419). Nearly all experimental rodent models of obesity are accompanied by diminished or defective BAT function, usually at the first symptom in the progression of obesity (Himms-Hagen, J. (1989) Prog. Lipid Res. 28:67-115; Himms-Hagen, J. (1990) FASEB J. 4:2890-2898). In addition, ablation of BAT in transgenic mice by targeted expression of a toxin gene results in obesity (Lowell, B. et al. (1993) Nature 366:740-742). Thus, the growth and differentiation of brown adipocytes are key determinants in an animal's ability to maintain energy balance and prevent obesity (Sears, I. B. et al. (1996) Mol. Cell. Biol. 16(7):3410-3419).
Recently, several transcription factors have been identified which promote adipogenesis. These transcription factors include CCAAT/enhancer binding protein (C/EBP) α, β, and δ and peroxisome proliferator activated receptor (PPAR) γ. See Spiegelman, B. M. and Flier, J. S. (1996) Cell 87:377-389 for a review. C/EBP family members such as C/EBPα, β, and δ play important roles in the regulation of adipocyte-specific gene expression. For example, C/EBPα can transactivate the promoters of several genes expressed in the mature adipocyte (Herrera, R. et al. (1989) Mol. Cell. Biol. 9:5331-5339; Miller, S. G. et al. (1996) PNAS 93:5507-551; Christy, R. J. et al. (1989) Genes Dev. 3:1323-1335; Umek, R. M. et al. (1991) Science 251:288-291; Kaestner, K. H. et al. (1990) PNAS 87:251-255; Delabrousse, F. C. et al. (1996) PNAS 93:4096-4101; Hwang, C. S. et al. (1996) PNAS 93:873-877). Overexpression of C/EBP α can induce adipocyte differentiation in fibroblasts (Freytag, S. O. et al. (1994) Genes Dev. 8:1654-1663) whereas expression of antisense C/EBPα inhibits terminal differentiation of preadipocytes (Lin, F. T and Lane, M. D. (1992) Genes Dev. 6:533-544). The physiological importance of C/EBPα was further demonstrated by the generation of transgenic, C/EBPα-knockout mice. Although adipocytes are still present in these animals, they accumulate much less lipid and exhibit decreased adipocyte-specific gene expression (Wang, N. et al. (1995) Science 269:1108-1112). C/EBPα was found to have a synergistic relationship with another transcription factor, PPARγ, in promoting adipocyte differentiation (See Brun, R. P. et al. (1996) Curr. Opin. Cell Biol. 8:826-832 for a review). PPARγ is a nuclear hormone receptor which exists in two isoforms (γ1 and γ2) formed by alternative splicing (Zhu, Y. et al. (1995) PNAS 92:7921-7925) and which appears to function as both a direct regulator of many fat-specific genes and also as a “master” regulator that can trigger the entire program of adipogenesis (Spiegelman, B. M. and Flier, J. S. (1996) Cell 87:377-389). PPARγ forms a heterodimer with RXRα and has been shown to bind directly to well characterized fat-specific enhancers from the adipocyte P2 (aP2: Tontonoz, P. (1994) Genes Dev. 8:1224-1234) and phosphoenolpyruvate carboxykinase (PEPCK) genes (Tontonoz, P. (1994) Mol. Cell. Biol. 15:351-357).
Although the UCP gene promoter includes binding sites for C/EBP (Yubero, P. et al. (1994) Biochem. Biophys. Res. Commun. 198:653-659) and a PPARγ-responsive element (Sears, I. B. et al. (1996) Mol. Cell. Biol. 16(7):3410-3419), C/EBP and PPARγ do not seem to be sufficient to induce UCP expression (Sears, I. B. et al. (1996) Mol. Cell. Biol. 16(7):3410-3419). It would be highly desirable, therefore, to identify a possible additional factor which acts in combination with either C/EBP or PPARγ to activate UCP expression and thus to promote BAT thermogenesis.